Many mosquito-borne viruses are associated with human diseases. Mosquitoes defend themselves against viral infection with an innate immune response. Thus, mosquito-borne viral diseases like West Nile fever, dengue fever, and chikungunya fever are transmitted to humans only when the pathogen is able to overcome these defenses. Despite this, relatively little is known about mosquito innate immunity. We have previously shown that an antiviral response directed by small interfering RNAs (siRNAs) is essential to the survival of mosquito vectors in the presence of an infecting mosquito-borne virus. However, more recently we demonstrated that another class of virus-derived small RNAs, exhibiting many similarities with ping-pong-dependent piwi- interacting RNAs (piRNAs), is also produced in the soma of mosquito vectors. We hypothesize that a non- canonical piRNA pathway present in the soma of vector mosquitoes is acting concurrently with the siRNA pathway to form a coordinated, redundant antiviral defense. In support of this, we characterized multiple mosquito cell lines defective in siRNA-based immunity, and then used these to show that a similar class of ping-pong-dependent viral piRNAs was capable of mounting an antiviral defense that controlled viral infection. In the first aim of this proposal, we will define the biognic pathway of viral piRNA production. In the second aim of the proposal, we will determine the antiviral role of the piRNA pathway in the mosquito soma. At the conclusion of this project, we will have a better understanding of mosquito antiviral immunity and the role of a non-canonical piRNA pathway in restricting virus infection. This information will help address gaps in our knowledge of the epidemiology of mosquito-borne viral diseases and also help to assess possibilities for broad spectrum control strategies that are based on manipulating an evolutionary arms race between viruses and their insect vectors.